菊欧文氏菌分子检测技术的研究

 蝴蝶兰细菌性软腐病对蝴蝶兰的生长危害严重, Erwinia chrysanthemi(菊欧文氏菌)、Erwinia carotovora subsp. carotovora(胡萝卜软腐欧文氏菌胡萝卜软腐亚种)是引起蝴蝶兰软腐病的主要病原细菌, 其中E.chrysanthemi被列入我国三类检疫性有害生物。本文对菊欧文氏菌分子检测技术进行了研究, 设计出针对该病原细菌的特异性引物, 应用实时荧光PCR方法检测样品中存在的菊欧文氏菌, 检测灵敏度达到10²cfu/mL。     

Bacterial Wilt of China Aster Caused by Erwinia chrysanthemi

In July 2000, a bacterial wilt was found on China aster (Callistephus chinensis Nees) on Hokkaido, Japan. Bacteriological characteristics of the isolated bacteria were identical to Erwinia chrysanthemi. This is the first report on bacterial wilt of China aster caused by E. chrysanthemi. Received 4 June 2001/ Accepted in revised form 6 September 2001     

Bacterial rot of witloof chicory roots caused by Erwinia chrysanthemi

Erwinia chrysanthemi (biovars 5 and 6) was isolated from unusual symptoms on witloof chicory, both in the field and in hydroponic culture, in Brittany in 1989 and 1990. Symptoms included a greyish-brown soft rot on the lower part of the root and the destruction of the cortical tissues. The cribrovascular and the medullary part of the bottom of the root sometimes became slimy. A few cases of vascular transmission were observed, which resulted in a red coloration of the infected vessels and a soft rot of the leaves.     

A new disease of cardamom (Elettaria cardamomum) caused by Erwinia chrysanthemi in Papua New Guinea

A serious disease of cardamom in East New Britain Province of Papua New Guinea was shown by pathogenicity and biochemical tests to be caused by Erwinia chrysanthemi. The bacterium was repeatedly isolated from blackened roots, soft discoloured rhizome tissue and rhizosphere soil, but not from field soil in the immediate neighbourhood of diseased plants.     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

Studies of a wilt disease of the potato plant in Israel caused by Erwinia chrysanthemi

In Israel field infections of potato plants by Erwinia chrysanthemi are characterized by wilting of the leaves followed by total desiccation of the plants. These symptoms are indistinguishable from those caused by Verticillium dahliae or those that develop during the normal process of plant senescence. Diagnosis of E. chrysanthemi in the spring-sown (February) crop in Israel is difficult because all three conditions often appear at approximately the same time, late in the growing season in May when the air temperature exceeds 25°C. The symptoms of E. chrysanthemi infection were reproduced in the field when potato seed tubers, tested and found to be contaminated at a low level with E. carotovora pv. carotovora , were inoculated with a strain of E. chrysanthemi isolated from a diseased potato plant. When plants in a growth cabinet at 30°C were stem-inoculated with E. chrysanthemi , similar symptoms developed when the relative humidity was low ( c . 80%). Presence of the disease only on plants grown from seed contaminated with E. chrysanthemi and not from uncontaminated seed suggests that the bacterium is seed borne, as is E. carotovora pv. atroseptica , the blackleg pathogen.     

一株对香蕉枯萎病菌具有良好拮抗作用的菊欧氏杆菌

在香蕉枯萎病重病园区,从生长正常香蕉假茎内分离获得一株细菌E353菌株。经对峙培养、孢子萌发抑制测定,E353对香蕉枯萎病菌菌丝生长、孢子萌发具有良好抑制效果。盆栽试验表明,E353活菌培养液（750ml／株）浸根处理,对香蕉枯萎病的防效为60．67％。经形态学、生理生化和16S rDNA序列比对,将E353鉴定为菊欧氏杆菌Erwinia chrysanthemi。     

Serological characterization of fluorescent Pseudomonas strains cross-reacting with antibodies against Erwinia chrysanthemi

Sixteen bacterial strains, cross-reacting with antibodies againstErwinia chrysanthemi (Ech), were isolated from potato peel extracts, ditch water, and the rhizosphere of wheat, onion, sugar beet and chicory using the immunofluorescence colony-staining procedure. Based on fatty acid profiles, isolates were classified as belonging to thePseudomonas fluorescens group.These strains, together with two previously isolated cross-reactingP. fluorescens strains, crossreacted with polyclonal antibodies against Ech in immunofluorescence cell-staining, Ouchterlony double diffusion, and ELISA. Seventeen strains also reacted strongly with monoclonal antibodies against the lipopolysaccharides (LPS) of Ech in ELISA.Cell envelopes (CE) and proteinase-K-treated CE (mainly LPS) of cross-reacting bacteria were further characterized with SDS-PAGE and Western blotting. Based on CE protein and LPS patterns, the cross-reacting bacteria were classified into two groups, each existing of two subgroups. Both CE and proteinase-K-resistant antigens strongly cross-reacted on immunoblots with antisera against a wild type strain of Ech. With an antiserum against a LPS O-chain lacking mutant of Ech only protein bands but no proteinase-K-resistant antigens were detected on immunoblots. These data suggest that in all cases the highly antigenic LPS O-chain is responsible for the cross-reactions.     

Detection of latent infection of Erwinia chrysanthemi and Pseudomonas caryophylli in carnation1

Three methods for detecting latent infections of Erwinia chrysanthemi and Pseudomonas caryophylli in carnation stems were studied. The same procedure was used, in all three methods, to prepare the samples and to extract and concentrate bacteria. The samples consisted of 100–500, 1-cm-long, pieces of carnation stems. Under the standard procedure, the samples were homogenized in maceration fluid, then centrifuged at low speed to eliminate the larger tissue fragments and centrifuged again at high speed to obtain a final pellet with a maximum concentration of the bacteria to be detected. Immunofluorescence staining (IFAS), direct isolation (DI) and immunoenzymatic test (ELISA) were all applied to the final pellets. IFAS had a sensitivity threshold equal to 8.65 × 10⁴ and 3.33 × 10⁴ bacteria per 100 stem pieces for E. chrysanthemi and P. caryophylli. In simulated latently infected lots IFAS could detect down to 0.2% infected pieces for both pathogens, both at high and at low infection level. Although DI had a similar theoretical sensitivity threshold for both bacteria, it was less successful when applied to simulated latently infected lots. ELISA had the highest theoretical sensitivity threshold for both bacteria and as a consequence could only detect them in simulated latently infected lots with a high infection level. The comparative results are discussed in relation to the applicability of the methods to commercial lots of carnation.     

Bacterial soft rot of myoga (Zingiber mioga) caused by Erwinia chrysanthemi

In 2010, a bacterial soft rot was found on myoga [Zingiber mioga (Thunb.) Rosc.] in Japan. Initial small spots on flower buds rapidly enlarged while changing from light brown to brown. Diseased flower buds became completely rotten. Several bacteria isolated from the diseased tissues caused the same symptoms as those observed in the fields and were reisolated from the tissue. These bacteria were identified as Erwinia chrysanthemi and were classified into subdivision IV and biovar 3. This is the first report of bacterial soft rot of myoga in the world.     

Infection of roots of Dieffenbachia maculata by the foliar blight and soft rot pathogen, Erwinia chrysanthemi

Erwinia chrysanthemi, the organism causing foliage blight, leaf spotting, basal stem rot and root rot of Dieffenbachia maculata, was shown to be capable of infecting via roots. Inoculated plants were sectioned and internal spread of the pathogen determined by 'sandwich' plating between two layers of Miller's selective medium. Light microscopy and SEM confirmed that the pathogen was present throughout the plant in the xylem. Resin ducts, previously claimed as being the route for systemic infection, were not found. Initially confined to the xylem, the pathogen spread intercellularly to the neighbouring parenchyma and, after about 8 days, pockets of infection, often surrounded by periderm–like tissues, were seen.     

The occurrence in Portugal of the bacterial disease of maize crops caused by Erwinia chrysanthemi

A bacterial disease causing stalk rot of maize was observed in Portugal, during the summer of 1991, in overhead sprinkler-irrigated fields. Affected plants showed symptoms of a soft rot disease and isolations made from infected tissues consistently yielded a bacterium. On the basis of morphological, cultural, biochemical, physiological and pathological properties, the pathogen was identified as Erwinia chrysanthemi. According to biovar discrimination tests, the maize isolates were allocated to biovars 2 and 3 of E. chrysanthemi.     

Characterization of Erwinia chrysanthemi, the soft-rot pathogen of white-flowered calla lily, based on pathogenicity and PCR-RFLP and PFGE analyses

Isolates of Erwinia chrysanthemi from Zantedeschia aethiopica (white-flowered calla lily) induced symptoms of soft rot on inoculated cv. Innocence flower-stem segments. Isolates from Phalaenopsis aphrodite and potato caused mild symptoms, while those from green onion and celery produced no symptoms. In addition to pathogenicity, the isolates were further characterized at the molecular level. A specific oligonucleotide primer set was designed for the detection of the pelZ gene of E. chrysanthemi . All E. chrysanthemi isolates tested contained pelZ as determined by PCR amplification. No amplification was observed with other Erwinia spp. The pelZ of E. chrysanthemi isolate S3-1 from Z. aethiopica was cloned, sequenced and compared with the nucleotide sequence of pelZ in GenBank. A point mutation produced an Ahd I restriction site, leading to the development of a PCR-RFLP assay to discriminate white-flowered calla lily isolates from others of E. chrysanthemi . Furthermore, macrorestriction analysis by modifying a pulsed-field gel electrophoresis (PFGE) protocol used by PulseNet revealed the genomic variation within E. chrysanthemi . After digestion with the restriction enzyme Xba I, white-flowered calla lily E. chrysanthemi isolates could be easily distinguished from other isolates. Differences in virulence, combined with PCR-RFLP and PFGE analyses, suggest that white-flowered calla lily E. chrysanthemi isolates are a new strain or pathotype in Taiwan.     

Identification of potential virulence genes in Erwinia chrysanthemi 3937: transposon insertion into plant-upregulated genes

Erwinia chrysanthemi 3937 is a soft-rotting plant pathogen in Enterobacteriaceae. It attacks a wide range of plant host species. Previously, we identified dozens of E. chrysanthemi 3937 genes induced during plant infection by microarray differential display. Here, we have mutated plant-upregulated and putatively plant-upregulated genes in E. chrysanthemi 3937 using a transposon insertion method. Of 57 mutants produced, 8 were significantly reduced in maceration in African violet leaves. These 8 E. chrysanthemi genes are similar to Escherichia coli purU (formyltetrahydrofolate deformylase; ASAP20623) and wcaJ (undecaprenylphosphate glucosephosphotransferase; ASAP18556), Bacillus subtilis dltA (d-alanine-d-alanyl carrier protein ligase; ASAP19406), Pseudomonas syringae PSPTO2912 (ABC transporter, periplasmic glutamine-binding protein; ASAP15639), Pseudomonas aeruginosa pheC (cyclohexadienyl dehydratase; ASAP19773), P. syringae syrE (peptide synthase; ASAP19989), Vibrio vulnificus VV12303 (unknown protein; ASAP18555), and Yersinia pestis speD (S-adenosylmethionine decarboxylase; ASAP20536). In some of the genes, possible roles in virulence could be postulated based on the functions of their homologues. This work demonstrated that a low proportion of pathogenicity-related genes were among the plant-upregulated genes of E. chrysanthemi 3937. This study and further dissection of these putative virulence genes should lead to new insights into infection mechanisms in pathogens.     

Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees

Bacteria from necrotic branches of Asian pear trees ( Pyrus pyrifolia ) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear ( Pyrus communis ) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia , but were different from Erwinia amylovora , the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2-D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora . No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP-PCR also produced bands differing for the two organisms.     

Emmer wheat, a potential new host of Tilletia indica

Two representative Italian emmer wheat (Triticum dicoccum) landraces, two selected lines and three improved emmer wheat cultivars, derived from crosses with durum wheat (Molisano landrace × ‘Simeto’), were tested for their susceptibility to Tilletia indica, the cause of Karnal bunt of wheat. Plants of emmer wheat were inoculated by injecting allantoid sporidial suspensions into the boot cavity of plants, just prior to ear emergence. A highly susceptible Indian spring wheat cultivar (Triticum aestivum) was used as a comparative control. At maturity of the plants, the seeds were harvested and assessed for incidence and severity of disease. All emmer wheat genotypes tested were infected but showed differing levels of susceptibility. The percentage of infected seeds for individual genotypes ranged from 5.4 to 75.0% compared with 99.1% for WL-711. The severity of infection was less in the old landraces, but it was higher in all the improved emmer wheat cultivars. In conclusion, Italian cultivars of emmer wheat were found to be highly susceptible to T. indica, and are potentially able to support the establishment of the pathogen.Authors L. Riccioni and M. Valvassori contributed equally to this work and should both be considered as first author.     

玉米细菌性茎腐病的发生为害调查

系统调查表明,玉米细菌性茎腐病由玉米细菌性茎腐病菌(Erwiniachrysanthemipv.zeae)所致,在玉米植株中部叶鞘和茎秆上发生水渍状腐烂,引起组织软化。病菌随病残组织在田间、地边越冬。夏季暴雨多、空气湿度大、虫害发生重等对病害发生有利。提出及时清除病残体、防治虫害、适时施药的防治措施。     

Ring rot-like symptoms in Solanum melongena caused by Erwinia chrysanthemi (potato strain) after artificial inoculation

Erwinia chrysanthemi , present in potato extracts prepared according to the EC method for detection of latent ring-rot infections, can multiply and cause ring rot-like symptoms in initial stages of disease development in the test plant Solanum melongena. Symptoms on S. melongena are described and differentiated from those caused by Clavibacter michiganensis ssp. sepedonicus.